This invention relates to seaweed extract compositions, enriched active fractions, isolated active agents, and methods of use for the treatment and/or prevention of reactive oxygen species (ROS)-mediated diseases and diseases alleviated or prevented through the activation of the Nrf2-ARE (antioxidant response element) pathway, such as inflammation, cancer, Alzheimer's disease and other neurodegenerative disorders, stroke, chronic kidney disease, type II diabetes, and aging itself.
In aerobes, reactive oxygen species (ROS) is produced during cellular respiration and energy metabolism [Halliwell, B. Biochemistry of oxidative stress. Biochem Soc Trans. 35:1147-50; 2007]. In a healthy cell, the level of ROS is tightly regulated by the antioxidant defense system. However, upon environmental stress or cellular damage, the cell cannot readily detoxify the ROS generated and may thereby suffer from oxidative stress, which is implicated in the pathogenesis of many age-related diseases, such as inflammation, cancer, Alzheimer's disease and other neurodegenerative disorders, stroke, chronic kidney disease, type II diabetes, and aging itself [Liu, Y.; Kern, J. T.; Walker, J. R.; Johnson, J. A.; Schultz, P. G.; Luesch, H. A genomic screen for activators of the antioxidant response element. Proc Natl Acad Sci USA. 104:5205-10; 2007; Dinkova-Kostova A T, Massiah M A, Bozak R E, Hicks R J, Talalay P. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci USA 2001; 98:3404-3409; Ramos-Gomez M, Kwak M-K, Dolan P M, Itoh K, Yamamoto M, Talalay P et. al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA 2001; 98:3410-3415; van Muiswinkel F L, Kuiperij H B. The Nrf2-ARE signaling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders. Curr Drug Targets CNS Neurol Disord 2005; 4:267-281; Dinkova-Kostova, A T, Liby K T, Stephenson K K, Holtzclaw W D, Gao X, Suh N et. al. Extremely potent triterpenoid inducers of the phase 2 response: Correlations of protection against oxidant and inflammatory stress. Proc Natl Acad Sci USA 2005; 102:4584-4589; Chen X-L, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr Pharm Des 2004; 10:879-891; Pergola P E, Raskin P, Toto R D, Meyer C J, Huff J W, Grossman E B et. al. BEAM Study Investigators. Bardoxolone methyl and kidney function in CKD with type 2 diabetes. N Engl J Med 2011; 365:327-336].
While antioxidant activity is commonly associated with direct radical scavenging activity, an alternative way to increase the antioxidant status of a cell or body is to concertedly enhance the endogenous defense system consisting of antioxidant enzymes and detoxification enzymes, which presumably causes a more sustained, longer-lasting effect. Phase II and other antioxidant enzymes are commonly regulated by the antioxidant response element (ARE) on the transcriptional level [Kensler T W, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47:89-116; 2007]. Increased expression of these enzymes correlates with a decrease in cellular damage by radical oxygen species (ROS), which are implicated in inflammation and the pathogenesis of many age-related disorders, including cancer, neurodegeneration, and aging itself [Chen X-L, Kunsch C. Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. Curr. Pharm. Des. 10:879-891; 2004; Surh Y J. Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer 3:768-780; 2003; van Muiswinkel F L, Kuiperij H B. The Nrf2-ARE signaling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders. Curr. Drug Targets CNS Neurol. Disord. 4:267-281; 2005]. In humans, the antioxidant response element (ARE) regulates the expression of cytoprotective antioxidant enzymes [e.g., heme oxygenase-1 (HO-1), glutathione-S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1)], which contribute to the endogenous defense against oxidative stress [Kensler T W, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47:89-116; 2007]. The major transcription factor involved in the induction of phase II enzymes is nuclear factor E2-related factor 2 (Nrf2), a Cap ‘n’ Collar (CNC) type basic region-leucine zipper (bZip) transcription factor that, upon activation by ARE inducers, translocates to the nucleus, binds to the ARE sequence as a heterodimer with one of the small bZip proteins, Mafs, and activates ARE-dependent genes. Nrf2 is negatively regulated by the cysteine-rich protein Keap1. Keap1 serves to sequester Nrf2 in the cytoplasm and interacts with Cul3-based E3 ubiquitin ligase to target Nrf2 for proteasomal degradation [Dinkova-Kostova A T, Holtzclaw W D, Kensler T W. The role of Keap1 in cellular protective responses. Chem. Res. Toxicol. 18:1779-1791; 2005; Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv. Enzyme Regul. 46:113-140; 2006; Zhang D D. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug. Metab. Rev. 38:769-789; 2006].
Nrf2 knockout mice show diminished detoxification capabilities, decreased responsiveness to chemoprotective agents, and enhanced susceptibility to oxidative stress induced cell death [Chan K, Han X-D, Kan Y W. An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc. Natl. Acad. Sci. USA 98:4611-4616; 2001; Ramos-Gomez M, Kwak M-K, Dolan P M, Itoh K, Yamamoto M, Talalay P, et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl. Acad. Sci. USA 98:3410-3415; 2001; Calkins M J, Jakel R J, Johnson D A, Chan K, Kan Y W, Johnson J A. Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc. Natl. Acad. Sci. USA 102:244-249; 2005]. Conversely, Nrf2 overexpression protects from oxidative stress [Chan K, Kan Y W, Johnson J A. Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc. Natl. Acad. Sci. USA 102:244-249; 2005]. NQO1-deficient individuals are at a considerably higher risk of developing leukemia following occupational exposure to benzene [Nebert D W, Roe A L, Vandale S E, Bingham E, Oakley G G. NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review. Genet. Med. 4:62-70; 2002]. The activation of the Nrf2-ARE pathway is a valid cancer preventive strategy, and sulforaphane, a constituent of broccoli, is an example of a cancer preventive natural product that acts through this mechanism [Surh Y J. Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer 3:768-780]. We hypothesized and preliminarily demonstrated that some seaweeds are able to activate this signaling pathway and that some of the beneficial, particularly antioxidant, properties may be mediated through ARE activation as opposed to only direct scavenging properties [Wang R, Paul V J, Luesch H. Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway. Free Rad. Biol. Med. doi10.1016/j.freeradbiomed.2012.12.019 (Epub Jan. 4, 2013); 2013].
ARE activation may also be particularly relevant to prostate cancer [Sikka S C. Role of oxidative stress response elements and antioxidants in prostate cancer pathobiology and chemoprevention—a mechanistic approach. Curr. Med. Chem. 10:2679-2692; 2003]. The most common hallmark in prostate cancer is the silencing of glutathione-S-transferase (GST)-π (GSTP1) due to DNA methylation, which is nearly universal [Lee W H, Morton R A, Epstein J I, Brooks J D, Campbell P A, Bova G S, et al. Cytidine methylation of regulatory sequences near the π-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA 91:11733-11737; 1994; Lee W-H, Isaacs W B, Bova G S, Nelson W G. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostatic biomarker. Cancer Epidemiol. Biomark. Prev. 6:443-450; 1997; Lin X, Tascilar M, Lee W H, Vies W J, Lee B H, Veeraswamy R, et al. GSTP1 cpG island hypermethylation is responsible for the absence of GSTP1 expression in human prostate cancer cells. Am. J. Pathol. 159:1815-1826; 2001]. Because of the lack of GSTP1 expression in prostate cancer (regardless of grade or stage), induction of GSTs and other phase II enzymes through ARE activation is a promising prostate cancer-preventive strategy [Lee W H, Morton R A, Epstein J I, Brooks J D, Campbell P A, Bova G S, et al. Cytidine methylation of regulatory sequences near the π-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA 91:11733-11737; 1994; Lee W-H, Isaacs W B, Bova G S, Nelson W G. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostatic biomarker. Cancer Epidemiol. Biomark Prev. 6:443-450; 1997; Lin X, Tascilar M, Lee W H, Vies W J, Lee B H, Veeraswamy R, et al. GSTP1 cpG island hypermethylation is responsible for the absence of GSTP1 expression in human prostate cancer cells. Am. J. Pathol. 159:1815-1826; 2001; Brooks J D, Paton V G, Vidanes G. Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol. Biomark. Prev. 10:949-954; 2001; Brooks J D, Goldberg M F, Nelson L A, Wu D, Nelson W G. Identification of potential prostate cancer preventive agents through induction of quinone reductase in vitro. Cancer Epidemiol. Biomark. Prev. 11:868-875; 2002]. While prostate cancer is the second leading cause of cancer death in American men, prostate cancer is rarely diagnosed and contributes little to cancer mortality in Asia [Greenlee R T, Hill-Harmon M B, Murray T, Thun M. Cancer statistics, 2001. CA Cancer J. Clin. 51:15-36; 2001; Carter B S, Carter H B, Isaacs J T. Epidemiologic evidence regarding predisposing factors to prostate cancer. Prostate 16:187-197; 1990; Yu H, Harris R E, Gao Y T, Gao R, Wynder E L. Comparative epidemiology of cancers of the colon, rectum, prostate, and breast in Shanghai, China versus the United States. Int. J. Epidemiol. 20:76-81; 1991]. However, men migrating from Asia to the USA increase their risk, which remains elevated in their male descendents [Shimizu H, Ross R K, Bernstein L, Yatani R, Henderson B E, Mack T M. Cancers of the prostate and breast among Japanese and white immigrants in Los Angeles County. Br. J. Cancer 63:963-966; 1991; Whittemore A S, Kolonel L N, Wu A H, John E M, Gallagher R P, Howe G R, et al. Prostate cancer in relation to diet, physical activity, and body size in blacks, whites, and Asians in the United States and Canada. J. Natl. Cancer Inst. 87:652-661; 1995; Haenzel W, Kurihara M, Mortality from cancer and other diseases among Japanese men in the United States. J. Natl. Cancer Inst. 40:43-68; 1968; Danley K L, Richardson J L, Bernstein L, Langholz B, Ross R K. Prostate cancer: trends in mortality and stage-specific incidence rates by racial/ethnic group in Los Angeles County, California (United States). Cancer Cause Control 6:492-498; 1995]. While environmental factors may play a role, this observation may be attributable to lifestyle changes. Notably, diet in Asia largely includes seaweed, suggesting a possible connection between algae consumption and decreased prostate cancer risk. Many other diseases, including those with an inflammation component, are caused by aberrant oxidative stress and may be prevented or interfered with via enhancing the cellular antioxidant status.
Marine algae (seaweeds) have been used as a food source and medicine for centuries [Chapman V J, Chapman D J. In Seaweeds and Their Uses. (Chapman and Hall, New York) pp 62-67; 1980]. This includes green algae (Chlorophyta), red algae (Rhodophyta) and brown algae (Ochrophyta). Consumption of seaweed, which predominantly occurs in Japan, was found to be inversely related to various cancers, including colon, rectal and stomach cancer [Hoshiyama Y, Sekine T, Sasaba T. A case-control study of colorectal-cancer and its relation to diet, cigarettes, and alcohol-consumption in Saitama Prefecture, Japan. Tohoku J. Exp. Med. 171:153-165; 1993; Hoshiyama Y, Sasaba T. A case-control study of single and multiple stomach cancers in Saitama Prefecture, Japan. Jpn. J. Cancer Res. 83:937-943; 1992]. Seaweed is a major part of the Okinawan food culture, and Okinawans have the longest life expectancy in the world and low disability rates [Sho H. History and characteristics of Okinawan longevity food. Asia Pac. J. Clin. Nutr. 10:159-164; 2001]. Numerous beneficial properties of algal extracts and constituents have been reported, however, usually only in a descriptive manner, without pinpointing specific bioactive components or invoking specific molecular pathways. Green algae of the genus Ulva, also known as sea lettuce, are among the most commonly consumed seaweeds. They reportedly have anti-inflammatory and antitumoral properties and are implicated in cancer prevention and detoxification. For example, crude extracts of U. reticulata given to rats attenuated acetaminophen-induced hepatotoxicity by improving the hepatic antioxidant status [Balaji Raghavendra Rao H, Sathivel A, Devaki T. Antihepatotoxic nature of Ulva reticulata (Chlorophycaeae) on acetaminophen-induced hepatotoxicity in experimental rats. J. Med. Food 7:495-497; 2004]. It has been postulated that Ulva extract protects the membrane from damage by toxic reactive metabolites produced by acetaminophen biotransformation [Balaji Raghavendra Rao H, Sathivel A, Devaki T. Antihepatotoxic nature of Ulva reticulata (Chlorophycaeae) on acetaminophen-induced hepatotoxicity in experimental rats. J. Med. Food 7:495-497; 2004]. U. conglobata has been found to have neuroprotective and anti-inflammatory activity, while U. lactuca has antitumor and immunostimulating effects [Jin D-Q, Lim C S, Sung J-Y, Choi H G, Ha I, Han J-S. Ulva conglobata, a marine algae, has neuroprotective and anti-inflammatory effects in murine hippocampal and microglial cells. Neurosci. Lett. 402:154-158; 2006; Lee D G, Hyun J W, Kang K A, Lee J O, Lee S H, Ha B J, et al. Ulva lactuca: a potential seaweed for tumor treatment and immune stimulation. Biotechnol. and Bioprocess E 9:236-238; 2004]. The antioxidant activity of U. pertusa has been attributed to polysaccharides with high sulfate content [Qi H, Zhang Q, Zhao T, Chen R, Zhang H, Niu X, et al. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. Int. J. Biol. Macromol. 37:195-199; 2005]. We recently described that U. lactuca can increase the cellular antioxidant status through an alternative mechanism, and attributed this activity in part to the presence of monounsaturated fatty acid constituents [Wang R, Paul V J, Luesch H. Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway. Free Rad. Biol. Med. doi10.1016/j.freeradbiomed.2012.12.019 (Epub Jan. 4, 2013); 2013].
Many naturally occurring small molecule inducers of the Nrf2-ARE pathway have been identified and explored as chemopreventive or therapeutic agents. For example, curcumin [Balogun, E.; Hogue, M.; Gong, P.; Killeen, E.; Green, C. J.; Foresti, R.; Alam, J.; Motterlini, R. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J. 371:887-95; 2003], the active ingredient in traditional herbal remedy and dietary spice turmeric (Curcuma longa) is currently in clinical trials for multiple conditions, including several cancers and Alzheimer's disease [Hatcher, H.; Planalp, R.; Cho, J.; Torti, F. M.; Torti, S. V. Curcumin: from ancient medicine to current clinical trials. Cell Mol Life Sci. 65:1631-52; 2008]. The skin of red grapes (Vitis vinifera) is rich in resveratrol [Langcake, P.; Pryce, R. J. Production of Resveratrol by Vitis-Vinifera and Other Members of Vitaceae as a Response to Infection or Injury. Physiological Plant Pathology. 9:77-86; 1976; Rubiolo, J. A.; Mithieux, G.; Vega, F. V. Resveratrol protects primary rat hepatocytes against oxidative stress damage: activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes. Eur J Pharmacol. 591:66-72; 2008], which was found to be responsible for an inverse relationship between grape consumption and breast cancer occurrence in an epidemiologic study [Levi, F.; Pasche, C.; Lucchini, F.; Ghidoni, R.; Ferraroni, M.; La Vecchia, C. Resveratrol and breast cancer risk. Eur J Cancer Prev. 14:139-42; 2005]. In a clinical setting, resveratrol was observed to induce the re-expression of tumor suppressor genes in a group of women who are at increased risk of breast cancer [Zhu, W.; Qin, W.; Zhang, K.; Rottinghaus, G. E.; Chen, Y. C.; Kliethermes, B.; Sauter, E. R. Trans-resveratrol alters mammary promoter hypermethylation in women at increased risk for breast cancer. Nutr Cancer. 64:393-400; 2012]. The detoxification enzyme inducer, sulforaphane [Kensler, T. W.; Egner, P. A.; Agyeman, A. S.; Visvanathan, K.; Groopman, J. D.; Chen, J. G.; Chen, T. Y.; Fahey, J. W.; Talalay, P. Keap1-Nrf2 Signaling: A Target for Cancer Prevention by Sulforaphane. Top Curr Chem. 2012], was found in many cruciferous vegetables. It has been shown that a daily regimen of hot water infused with 3-day-old broccoli sprouts has promising results in cancer chemoprevention in healthy individuals [Kensler, T. W.; Chen, J. G.; Egner, P. A.; Fahey, J. W.; Jacobson, L. P.; Stephenson, K. K.; Ye, L.; Coady, J. L.; Wang, J. B.; Wu, Y.; Sun, Y.; Zhang, Q. N.; Zhang, B. C.; Zhu, Y. R.; Qian, G. S.; Carmella, S. G.; Hecht, S. S.; Benning, L.; Gange, S. J.; Groopman, J. D.; Talalay, P. Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township, Qidong, People's Republic of China. Cancer Epidemiol Biomarkers Prev. 14:2605-13; 2005]. Broccoli sprouts (Brassica oleracea italica) contain high levels of its precursor, glucoraphanin [Farnham, M. W.; Stephenson, K. K.; Fahey, J. W. Glucoraphanin level in broccoli seed is largely determined by genotype. Hortscience. 40:50-53; 2005], which can be enzymatically converted to sulforaphane in the gastrointestinal tract after ingestion [Zhang, Y.; Talalay, P.; Cho, C. G.; Posner, G. H. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA. 89:2399-403; 1992].
The marine environment has also proven to be a rich source of potent compounds with diverse therapeutic properties [Newman, D. J.; Cragg, G. M. Marine natural products and related compounds in clinical and advanced preclinical trials. J Nat Prod. 67:1216-38; 2004; Montaser, R.; Luesch, H. Marine natural products: a new wave of drugs? Future Med Chem. 3:1475-89; 2011]. For example, several molecules with anti-cancer activities based on leads from marine cyanobacteria have been described [Taori, K.; Paul, V. J.; Luesch, H. Structure and activity of largazole, a potent antiproliferative agent from the Floridian marine cyanobacterium Symploca sp. J Am Chem Soc. 130:1806-7; 2008-20; Hong, J.; Luesch, H. Largazole: from discovery to broad-spectrum therapy. Nat Prod Rep. 29:449-56; 2012; Chen, Q. Y.; Liu, Y.; Luesch, H. Systematic Chemical Mutagenesis Identifies a Potent Novel Apratoxin A/E Hybrid with Improved in Vivo Antitumor Activity. ACS Med Chem Lett. 2:861-865; 2011]. Additionally, the free radical scavenger fucoxanthin, a carotenoid from a common edible seaweed, Hijikia fusiformis [Yon, X.; Chuda, Y.; Suzuki, M.; Nagata, T. Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Biosci Biotechnol Biochem. 63:605-7; 1999], was found to activate the antioxidant defense system (Nrf2/ARE) in mouse liver cells.
However, despite these developments, there exists an unmet need for additional antioxidants and for additional treatments for ROS-mediated diseases. This study follows on previous research that showed that extracts of marine algae can activate the Nrf2-ARE pathway, and that extracts of Ulva spp. were particularly active among a variety of seaweeds tested [Wang R, Paul V J, Luesch H. Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway. Free Rad. Biol. Med. doi10.1016/j.freeradbiomed.2012.12.019 (Epub Jan. 4, 2013); 2013]. As a result of ongoing investigations to identify new drug leads from marine sources, we report seaweed extract compositions isolated from cultivated green alga Ulva sp., processes for isolation, enriched active fractions, and isolated active agents. The extracts, enriched active extracts, and compounds herein are found to be activators of the cytoprotective Nrf2-ARE pathway. These findings provide new alternatives for the treatment and/or prevention of reactive oxygen species (ROS)-mediated diseases and diseases alleviated or prevented through the activation of the Nrf2-ARE (antioxidant response element) pathway, such as inflammation, cancer, Alzheimer's disease and other neurodegenerative disorders, stroke, chronic kidney disease, type II diabetes, and aging itself.